Molecular typing of Cryptococcus neoformans: taxonomic and epidemiological aspects.

Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, RAPD patterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.

Involvement of mitochondria in the assimilatory metabolism of anaerobic Saccharomyces cerevisiae cultures.

The possible physiological role of mitochondria in anaerobically grown Saccharomyces cerevisiae was investigated via enzyme localization and inhibitor studies. Almost all of the activity of citrate synthase (EC 4.1.3.7) was recovered in the mitochondrial fraction after differential centrifugation of spheroplast lysates. The enzyme exhibited a high degree of latency which was demonstrated by sonication of the mitochondrial fractions. Since citrate synthase is an important enzyme in anabolic reactions, a consequence of this localization is the requirement for transport of metabolites across the mitochondrial membranes. Such transport is likely to require energy which, as a result of anaerobiosis, cannot be supplied by respiration. It was therefore investigated whether ATP translocation into the mitochondria by an ADP/ATP translocase might be involved in anaerobic mitochondrial energy metabolism. It was shown that addition of the ADP/ATP translocase inhibitor bongkrekic acid to anaerobic cultures indeed inhibited growth, although only partially. It is concluded that mitochondria of S. cerevisiae fulfil a vital role in anaerobic sugar metabolism.

Is the Kluyver effect in yeasts caused by product inhibition?

Candida utilis CBS 621 exhibits the Kluyver effect for maltose, i.e. this yeast can respire maltose and is able to ferment glucose, but is unable to ferment maltose. When glucose was pulsed to a maltose-grown, oxygen-limited chemostat culture of C. utilis, ethanol formation from glucose started almost instantaneously, indicating that the enzymes needed for alcoholic fermentation are expressed in maltose-grown cells. However, the addition of glucose inhibited maltose metabolism. To eliminate a possible catabolite inhibition and/or repression of enzyme activities involved in maltose metabolism, the effect of simultaneously feeding glucose and maltose to an oxygen-limited, maltose-grown chemostat culture was studied. In this case, the glucose concentration in the culture remained below 0.1 mM, which makes glucose catabolite repression unlikely. Nevertheless, maltose metabolism appeared to cease when the culture was switched to the mixed feed. Based on the outcome of the mixed-substrate studies, it was postulated that the Kluyver effect may be caused by feedback inhibition of maltose utilization by ethanol, the product of fermentative maltose metabolism. If ethanol suppresses the utilization of non-fermentable disaccharides, this would provide a phenomenological explanation for the occurrence of the Kluyver effect: accumulation would then not occur and the rate of maltose metabolism would be tuned to the culture's respiratory capacity. This hypothesis was tested by studying growth of C. utilis CBS 621 and Debaryomyces castellii CBS 2923 in aerobic batch cultures on mixtures of sugars and ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of oxygen limitation on sugar metabolism in yeasts: a continuous-culture study of the Kluyver effect.

Growth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0.1 h-1. With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol l-1 h-1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0.1 h-1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable: alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.

Propionate metabolism in Saccharomyces cerevisiae: implications for the metabolon hypothesis.

Aerobic, glucose-limited chemostat of Saccharomyces cerevisiae CBS 8066 co-metabolized propionate when this compound was added to the reservoir medium. Co-metabolism of propionate led to an increase of the biomass and protein yields. Attempts to grow S. cerevisiae on propionate as a sole source of carbon and energy were not successful. Activities of propionyl-CoA synthetase in cell-free extracts were sufficient to account for the rates of propionate consumption observed in the chemostat cultures. Activities of propionyl-CoA carboxylase, a key enzyme of the methylmalonyl-CoA pathway of propionate metabolism, were negligible. In contrast, activities of 2-methylcitrate synthase, a key enzyme activity of the 2-methylcitrate pathway of propionate metabolism, increased substantially with increasing propionate-to-glucose ratios in the reservoir media, and were sufficient to account for the propionate consumption rates observed in the chemostat cultures. This suggested that the 2-methylcitrate pathway is the major pathway of propionate metabolism in S. cerevisiae. In the literature, labelling patterns observed after incubation of this yeast with [3-13C]propionate have been interpreted as evidence for channelling of tricarboxylic acid (TCA) cycle intermediates, possibly as a consequence of the organization of TCA cycle enzymes in a metabolon. However, this interpretation of 13C-labelling patterns rested on the assumption that propionate metabolism in S. cerevisiae occurs via the methylmalonyl-CoA pathway. Since the distribution of 13C in alanine reported in the literature is fully compatible with a major role of the 2-methylcitrate pathway in propionate metabolism, it cannot be interpreted as evidence for the existence of a TCA cycle metabolon in S. cerevisiae.

Energetic aspects of glucose metabolism in a pyruvate-dehydrogenase-negative mutant of Saccharomyces cerevisiae.

Saccharomyces cerevisiae T23C (pda1::Tn5ble) is an isogenic gene replacement mutant of the wild-type strain S. cerevisiae T23D. The mutation causes a complete loss of pyruvate dehydrogenase activity. Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh-) strain was investigated in aerobic glucose-limited chemostat cultures, grown at a dilution rate of 0.10 h-1, and compared with the metabolism in the isogenic wild-type strain. Under these conditions, growth of the Pdh- strain was fully respiratory. Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass of the pyruvate dehydrogenase complex. Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the pda1::Tn5ble mutation on the growth efficiency was anticipated. Indeed, the biomass yield of the Pdh- strain [0.44 g biomass (g glucose)-1] was significantly lower than that of wild-type S. cerevisiae [0.52 g biomass (g glucose)-1]. The effect of the mutation on biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism. Control experiments showed that the pda1::Tn5ble mutation did not affect biomass yield in ethanol-limited chemostat cultures. The results support the view that, during aerobic glucose-limited growth of S. cerevisiae at low growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux. Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in this yeast. Respiratory-deficient cells did not contribute to glucose metabolism in the chemostat cultures and were probably formed upon plating.

Energetics and kinetics of maltose transport in Saccharomyces cerevisiae: a continuous culture study.

In Saccharomyces cerevisiae, maltose is transported by a proton symport mechanism, whereas glucose transport occurs via facilitated diffusion. The energy requirement for maltose transport was evaluated with a metabolic model based on an experimental value of YATP for growth on glucose and an ATP requirement for maltose transport of 1 mol.mol-1. The predictions of the model were verified experimentally with anaerobic, sugar-limited chemostat cultures growing on a range of maltose-glucose mixtures at a fixed dilution rate of 0.1 h-1. The biomass yield (grams of cells.gram of sugar-1) decreased linearly with increasing amounts of maltose in the mixture. The yield was 25% lower during growth on maltose than during that on glucose, in agreement with the model predictions. During sugar-limited growth, the residual concentrations of maltose and glucose in the culture increased in proportion to their relative concentrations in the medium feed. From the residual maltose concentration, the in situ rates of maltose consumption by cultures, and the Km of the maltose carrier for maltose, it was calculated that the amount of this carrier was proportional to the in situ maltose consumption rate. This was also found for the amount of intracellular maltose. These two maltose-specific enzymes therefore exert high control over the maltose flux in S. cerevisiae in anaerobic, sugar-limited, steady-state cultures.

The use of karyotyping in the systematics of yeasts.

The use of electrophoretic karyotyping in systematics of yeasts is discussed. New data are provided on the karyotypes of the medically important fungi Hortaea werneckii, Filobasidiella (= Cryptococcus) neoformans, and Malassezia species. Hortaea werneckii has twelve to eighteen bands of chromosomal DNA, ranging in size between 500 and 2300 kb. The karyotypes of Filobasidiella neoformans consist of seven to fourteen bands of chromosomal DNA. The varieties neoformans and bacillispora cannot be separated by their karyotypes, and no obvious correlation was found with serotypes, geography or habitat. All strains of Malassezia pachydermatis studied have similar karyotypes consisting of five bands, whereas in M. furfur, four different karyotypes are prevalent. However, each of these karyotypes is stable.

Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation.

Addition of benzoate to the medium reservoir of glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h-1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g-1 h-1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in qO2 to 13 mmol g-1 h-1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash-out. As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest to study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D = 0.10 h-1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the extracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate. Growth of S. cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1 h-1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume. Also with the Crabtree-negative yeasts Candida utilis, Kluyveromyces marxianus and Hansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S. cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h-1 in the presence of benzoate was equal to, or lower than the qO2 attainable at mu(max) without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K. marxianus when the glucose flux was increased by the presence of benzoate.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of protein concentration by total organic carbon analysis.

Determination of the carbon concentration in protein solutions by total organic carbon analysis was found to be a sensitive and reliable method for the estimation of protein concentrations. Using a carbon content of 0.53 g/g in protein and of 0.44 g/g in carbohydrate, the concentrations of normal proteins, proteins containing chromophoric groups, and proteins containing carbohydrate could be established. The method appeared to be independent of the nature of the protein and showed complete linearity between 25 and 1000 mg/l (0.5-20 micrograms per assay) when protein was serially diluted. Determination of specific absorption coefficients by measuring both the absorbance of protein solutions at 280 nm and their carbon concentrations gave values which, on the average, coincided within 12% with values reported in the literature. The method may have special applicability in protein purification studies, as it does not require knowledge of molar extinction coefficients beforehand, and also monitors the disappearance of carbon compounds other than protein.

Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts.

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.

Hydrogen peroxide as an electron acceptor for mitochondrial respiration in the yeast Hansenula polymorpha.

Chemostat cultures of a catalase-negative mutant of Hansenula polymorpha CBS 4732 were able to decompose hydrogen peroxide at a high rate. This was apparent from experiments in which the yeast was grown under carbon limitation in chemostat culture on mixtures of glucose and H2O2. The enzyme responsible for H2O2 degradation is probably the mitochondrial enzyme cytochrome c peroxidase (CCP), which was present at very high activities. This enzyme was partially purified and shown to be specific for reduced cytochrome c as an electron donor; no reaction was observed with NAD(P)H. Thus, reducing equivalents for H2O2 degradation by CCP must be provided by the respiratory chain. That H2O2 can act as an electron acceptor for reducing equivalents could be confirmed with experiments in which cells were incubated with ethanol and H2O2 in the absence of oxygen. This resulted in oxidation of ethanol to equimolar amounts of acetate. Energetic aspects of mitochondrial H2O2 decomposition via CCP and the physiological function of CCP in yeasts are discussed.

A theoretical evaluation of growth yields of yeasts.

Growth yields of Saccharomyces cerevisiae and Candida utilis in carbon-limited chemostat cultures were evaluated. The yields on ethanol and acetate were much lower in S. cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast. However, during aerobic growth on glucose both organisms had the same cell yield. This can be attributed to two factors: --S. cerevisiae had a lower protein content than C. utilis; --uptake of glucose by C. utilis requires energy whereas in S. cerevisiae it occurs via facilitated diffusion. Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation in C. utilis is 35% higher than in S. cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively). The experimental YATP for anaerobic growth of S. cerevisiae on glucose was 16 g biomass.mol ATP-1. In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account. This was done in two ways: --via the assumption that the gap is independent of the growth substrate (i.e. a fixed amount of ATP bridges the difference between the theoretical and experimental values). --alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate. Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate. These P/O-ratios are approximately 30% lower than the calculated mechanistic values.

Oxygen requirements of yeasts.

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.

Localization of inulinase and invertase in Kluyveromyces species.

In vivo hydrolysis of inulin and sucrose was examined in selected yeasts of the genus Kluyveromyces. Cells, grown in sucrose-limited chemostat cultures, were subjected to treatments for the removal of inulinase, the enzyme responsible for the hydrolysis of both inulin and sucrose. The effects of these treatments were studied by measurement of inulin-dependent and sucrose-dependent oxygen consumption by cell suspensions. In Kluyveromyces marxianus var. marxianus, inulinase was partially secreted into the culture fluid. Removal of culture fluid inulinase by washing had no effect on sucrose-dependent oxygen consumption by this yeast. However, this treatment drastically reduced inulin-dependent oxygen consumption. Treatment of washed cells with sulfhydryls removed part of the cell wall-retained inulinase and reduced inulin-dependent oxygen consumption by another 80%. Sucrose-dependent oxygen consumption was less affected, decreasing by 40%. Cell suspensions of K. marxianus var. drosophilarum, K. marxianus var. vanudenii, and Saccharomyces kluyveri rapidly utilized sucrose but not inulin. This is in accordance with the classification of these yeasts as inulin negative. Supernatants of cultures grown at pH 5.5 did not catalyze the hydrolysis of inulin and sucrose. This suggested that these yeasts contained a strictly cell-bound invertase, an enzyme not capable of inulin hydrolysis. However, upon washing, cells became able to utilize inulin. The inulin-dependent oxygen consumption further increased after treatment of the cells with sulfhydryls. These treatments did not affect the sucrose-dependent oxygen consumption of the cells. Apparently, these treatments removed a permeability barrier for inulin that does not exist for sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556.

In the yeast Kluyveromyces marxianus two forms of inulinase were present, namely, an inulinase secreted into the culture fluid and an inulinase retained in the cell wall. Both forms were purified and analyzed by denaturing and nondenaturing polyacrylamide gel electrophoresis. With the use of endo-beta-N-acetyl-glucosaminidase H, it was established that the enzyme retained in the cell wall and the enzyme secreted into the culture fluid have similar subunits consisting of a 64-kDa polypeptide with varying amounts of carbohydrate (26 to 37% of the molecular mass). The two forms of inulinase differed in size because of their differences in subunit aggregation. The enzyme present in the culture fluid was a dimer, and the enzyme retained in the cell wall was a tetramer. The differences in oligomerization did not affect the apparent Km values towards the substrates sucrose and raffinose. These findings support the hypothesis that the retention of glycoproteins in the yeast cell wall may be caused by a permeability barrier towards larger glycoproteins. The amino-terminal end of inulinase was determined and compared with the amino terminus of the closely related invertase. The kinetic and structural evidence indicates that in yeasts two distinct beta-fructosidases exist, namely, invertase and inulinase.

Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures.

The energetics of Saccharomyces cerevisiae were studied in anaerobic glucose-limited chemostat cultures via an analysis of biomass and metabolite production. The observed YATP was dependent on the composition of the biomass, the production of acetate, the extracellular pH, and the provision of an adequate amount of fatty acid in the medium. Under optimal growth conditions, the YATP was approximately 16 g biomass (mol ATP formed)-1. This is much higher than previously reported for batch cultures. Addition of acetic acid or propionic acid lowered the YATP. A linear correlation was found between the energy required to compensate for import of protons and the amount of acid added. This energy requirement may be regarded as a maintenance energy, since it was independent of the dilution rate at a given acid concentration.

Substrate-accelerated death of Saccharomyces cerevisiae CBS 8066 under maltose stress.

When Saccharomyces cerevisiae CBS 8066 was grown under maltose limitation, two enzymes specific for maltose utilization were present: a maltose carrier, and the maltose-hydrolysing alpha-glucosidase. The role of these two enzymes in the physiology of S. cerevisiae was investigated in a comparative study in which Candida utilis CBS 621 was used as a reference organism. Maltose pulses to a maltose-limited chemostat culture of S. cerevisiae resulted in 'substrate-accelerated death'. This was evident from: (1) enhanced protein release from cells; (2) excretion of glucose into the medium; (3) decreased viability. These effects wee specific with respect to both substrate and organism: pulses of glucose to maltose-limited cultures of S. cerevisiae did not result in cell death, neither did maltose pulses to maltose-limited cultures of C. utilis. The maltose-accelerated death of s. cerevisiae is most likely explained in terms of an uncontrolled uptake of maltose into the cell, resulting in an osmotic burst. Our results also provide evidence that the aerobic alcoholic fermentation that occurs after pulsing sugars to sugar-limited cultures of s. cerevisiae (short-term Crabtree effect) cannot solely be explained in terms of the mechanism of sugar transport. Both glucose and maltose pulses to maltose-limited cultures triggered aerobic alcohol formation. However, glucose transport by S. cerevisiae occurs via facilitated diffusion, whereas maltose entry into this yeast is mediated by a maltose/proton symport system.

Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80. The organism had a mu max of 0.31 h-1 and a Ks for glucose of 0.55 mM. At a dilution rate of 0.10 h-1, a maximal yield of 0.10 g biomass (g glucose)-1 was observed. The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 0.10 h-1, the yield of the S. cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition. The major difference between the two yeast strains was that S. cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid. The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase. The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed. This resulted in a reduction in yield and an increase in specific ethanol production. Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth. Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.

Transient-state analysis of metabolic fluxes in crabtree-positive and crabtree-negative yeasts.

In bakers' yeast, an immediate alcoholic fermentation begins when a glucose pulse is added to glucose-limited, aerobically grown cells. The mechanism of this short-term Crabtree effect was investigated via a comparative enzymic analysis of eight yeast species. It was established that the fermentation rate of the organisms upon transition from glucose limitation to glucose excess is positively correlated with the level of pyruvate decarboxylase (EC 4.1.1.1). In the Crabtree-negative yeasts, the pyruvate decarboxylase activity was low and did not increase when excess glucose was added. In contrast, in the Crabtree-positive yeasts, the activity of this enzyme was on the average sixfold higher and increased after exposure to glucose excess. In Crabtree-negative species, relatively high activities of acetaldehyde dehydrogenases (EC 1.2.1.4 and EC 1.2.1.5) and acetyl coenzyme A synthetase (EC 6.2.1.1), in addition to low pyruvate decarboxylase activities, were present. Thus, in these yeasts, acetaldehyde can be effectively oxidized via a bypass that circumvents the reduction of acetaldehyde to ethanol. Growth rates of most Crabtree-positive yeasts did not increase upon transition from glucose limitation to glucose excess. In contrast, the Crabtree-negative yeasts exhibited enhanced rates of biomass production which in most cases could be ascribed to the intracellular accumulation of reserve carbohydrates. Generally, the glucose consumption rate after a glucose pulse was higher in the Crabtree-positive yeasts than in the Crabtree-negative yeasts. However, the respiratory capacities of steady-state cultures of Crabtree-positive yeasts were not significantly different from those of Crabtree-negative yeasts. Thus, a limited respiratory capacity is not the primary cause of the Crabtree effect in yeasts. Instead, the difference between Crabtree-positive and Crabtree-negative yeasts is attributed to differences in the kinetics of glucose uptake, synthesis of reserve carbohydrates, and pyruvate metabolism.